Increased accuracy in identifying NAFLD with advanced fibrosis and cirrhosis: independent validation of the Agile 3+ and 4 scores

BACKGROUND AND AIMS
We explored 2 novel scores, Agile 3+ and 4, to identify advanced fibrosis (≥F3) and cirrhosis (F4), respectively, in NAFLD and compared their diagnostic performances to liver stiffness measurement (LSM) by vibration-controlled transient elastography and fibrosis-4 index (FIB-4) (for Agile 3+).


APPROACH AND RESULTS
This multicenter study included 548 NAFLD patients with laboratory testing, liver biopsy, and vibration-controlled transient elastography within 6 months. Agile 3+ and 4 were applied and compared with FIB-4 or LSM alone. Goodness of fit was evaluated using a calibration plot and discrimination using area under the receiver operating curve. Area under the receiver operating curves was compared using the Delong test. Dual cutoff approaches were applied to rule out and rule in ≥F3 and F4. Median (interquartile range) age was 58 (15) years. Median body mass index was 33.3 (8.5) kg/m2. Fifty-three percent had type 2 diabetes, 20% had F3, and 26% had F4. Agile 3+ demonstrated an area under the receiver operating curve of 0.85 (0.81; 0.88) similar to that of LSM [0.83 (0.79; 0.86), p=0.142] but significantly higher than that of FIB-4 [0.77 (0.73; 0.81), p<0.0001). Agile 4's area under the receiver operating curve [0.85 (0.81; 0.88)] was similar to that of LSM [0.85 (0.81; 0.88), p=0.065). However, the percentage of patients with indeterminate results was significantly lower with Agile scores compared with FIB-4 and LSM (Agile 3+: 14% vs. FIB-4: 31% vs. LSM: 13%, p<0.001; Agile 4: 23% vs. LSM: 38%, p<0.001).


CONCLUSIONS
Agile 3+ and 4 are novel vibration-controlled transient elastography-based noninvasive scores that increase accuracy in the identification of advanced fibrosis and cirrhosis respectively and are ideal for clinical use due to a lower percentage of indeterminant outputs compared with FIB-4 or LSM alone.

Conclusions: Agile 3+ and 4 are novel vibration-controlled transient elastography-based noninvasive scores that increase accuracy in the identification of advanced fibrosis and cirrhosis respectively and are ideal for clinical use due to a lower percentage of indeterminant outputs compared with FIB-4 or LSM alone.
NAFLD is the most common cause of chronic liver disease worldwide, affecting 25%-35% of the global adult population and up to 70% of those with type 2 diabetes and obesity. [1] Because NASH is associated with fibrosis, cirrhosis, and even HCC, NAFLD is one of the leading indicators for liver transplantation in Europe and the US. [1,2] As NAFLD is often clinically silent, a key challenge is identifying those with advanced fibrosis (fibrosis stage of ≥ F3) and cirrhosis (F4) who are at significantly higher risk of liver-related mortality. [3] Currently, risk stratification of liver fibrosis ranges from noninvasive assessment scores to percutaneous liver biopsy. Although the reference method to assess liver fibrosis, liver biopsy is invasive and limited by cost, sampling variability, and intrareader/interreader variability. [4] Noninvasive modalities to risk stratify fibrosis include serum biomarkers, imaging, and algorithms combining both. Among the serum biomarkers, fibrosis-4 index (FIB-4) and NAFLD fibrosis score have demonstrated high accuracy in excluding advanced fibrosis with negative predictive values > 90%. [5] In regard to imaging modalities, liver stiffness measurement (LSM) by vibration-controlled transient elastography (VCTE) is accurate in excluding advanced fibrosis and cirrhosis with negative predictive values of~90%. [6] However, FIB-4, NAFLD fibrosis score, and LSM by VCTE are inadequate for ruling in advanced fibrosis or cirrhosis and often necessitate additional testing in the case of positive results. [7] The novel Fibroscan-based Agile 3+ and 4 scores combining LSM by VCTE with constitutive demographic data (age, sex, and presence of type 2 diabetes) and serum biomarkers (aspartate aminotransferase, alanine transaminase, and platelets) were recently introduced to better rule in advanced fibrosis and cirrhosis, respectively, in NAFLD. [8,9] We aimed to perform an independent validation of the Agile 3+ and 4 scores performance for diagnosing advanced fibrosis and cirrhosis, respectively, in a mulicenter real-world cohort of NAFLD patients in the US. We further compared the Agile 3+ and 4 score performances to FIB-4 and LSM using previously published cutoff values.

Participants
This analysis included adults with biopsy-proven NAFLD from 1 outpatient clinic and 3 tertiary care centers from Arizona (Arizona Liver Health), California (Cedars Sinai Medical Center, California Liver Institute), and Indiana (Indiana University) in the US.
Data were collected between 2014 and 2021 from patients with NAFLD who underwent liver biopsy, VCTE, and laboratory tests within 6 months. Subjects were excluded for the following: (1) missing data necessary for calculating the Agile 3+ and 4 scores, (2) missing fibrosis stage on liver biopsy, or (3) history of chronic liver disease other than NAFLD including autoimmune hepatitis, α1-antitrypsin deficiency, chronic hepatitis B or C, primary sclerosing cholangitis, primary biliary cholangitis, hemochromatosis, Wilson disease, or medications that can drive hepatic steatosis. Patients completed in-depth medical history, physical examination, and laboratory assessment before undergoing LSM. Liver biopsy was performed in the presence of abnormalities that raised concern for clinically significant NAFLD such as high LSM or diabetes, with or without elevated liver biochemistries. This study was exempt from institutional review board.

Vibration-controlled transient elastography
For LSM by VCTE (Fibroscan 502 Touch, Echosens, Paris, France), the speed of a mechanically generated 50 Hz shear wave across the liver was measured and then converted into LSM in kilo Pascals (kPa). VCTE was performed on patients after fasting for at least 3 hours before the examination by certified physicians, nurses, or technicians who were blinded to clinical data. The M or XL probe was selected based on the automatic probe selection tool provided by the software. After patients were placed supine with the fully abducted right arm, the right liver lobe was scanned through an intercostal space to obtain a minimum of 10 valid measurements. The final results consisted of the median of all valid measurements and was considered adequate for statistical analysis if the interquartile range over the median ratio was inferior or equal to 30%. [10] Liver histology The histological assessments at the local institutions were performed by hepatopathologists according to the NASH Clinical Research Network scoring system. [11] Fibrosis stage (F0-F4) was defined as no fibrosis (F0), either mild-moderate perisinusoidal or periportal fibrosis (F1), both perisinusoidal and portal/periportal fibrosis (F2), bridging fibrosis (F3), and cirrhosis (F4).

Outcomes
The main outcomes were the performance of Agile 3+ and 4 for the diagnosis of advanced fibrosis ( ≥ F3) and cirrhosis (F4), respectively.

Statistical analysis
Patient characteristics were reported as median (interquartile range) and the number of available data for numerical variables and frequency and percentage for categorical variables. For each patient, Agile 3+ and Agile 4 were calculated as: Considering diabetes status: yes = 1, no = 0 and sex: male = 1, female = 0, Both scores' performances were assessed using fibrosis stage by histology as the reference by the goodness of fit and discrimination and compared with LSM alone and FIB-4 used as predictors of advanced fibrosis and cirrhosis. The goodness of fit (the agreement between observed outcome and prediction) was evaluated using calibration plots and discrimination using the area under the receiver operating curve (AUROC). [12] AUROC comparisons were performed using the Delong test (at a 2sided 5% significance level). [13] Dual cutoff approach with cutoffs of high sensitivity and high specificity already published for the different noninvasive tests was applied to rule out and rule in advanced fibrosis and cirrhosis. [8,9] When appraising performance at a given cutoff, sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio and negative likelihood ratio were computed. Proportion of patients with indeterminate results with each noninvasive method were compared using z-test. Statistical analyses were computed using the R software (https://www.r-project.org/)

Agile 4 performance in comparison to LSM for the identification of cirrhosis
The performance of Agile 4 in identifying those with cirrhosis (F4) compared with LSM is shown in Figure 4 and Table 3

DISCUSSION
Given the clinical silence of NAFLD disease progression, it is essential to identify those with advanced fibrosis and cirrhosis, as later stages of fibrosis are associated with higher risk of mortality and necessitate therapeutic intervention. [3] This study independently validated the Agile 3+ and 4 scores for noninvasively identifying NAFLD patients with advanced fibrosis and cirrhosis, respectively, and compared the Agile 3+ and 4 scores' performances to those of FIB-4 and LSM.
Both the Agile 3+ and 4 scores performed superiorly to FIB-4 and LSM by VCTE and maintained good accuracy for the diagnosis of advanced fibrosis and cirrhosis, respectively. Both the Agile 3+ and 4 scores demonstrated good calibration with the curve close to the ideal calibration line. Both Agile 3+ and 4 scores exhibited good accuracy, though their AUROCs were not significantly different but numerically higher from those of LSM. The Agile 3+ and 4 scores' strengths lie in their 2-score cutoffs approach that significantly decreased the percentages of patients in the indeterminate zone compared with FIB-4 and LSM. The Agile scores demonstrated (1) improvement in the number of patients properly ruled out with higher specificity and negative predictive value in the rule-out zone for both Agile 3+ and 4 compared with FIB-4 and LSM except for similar specificity to that of FIB-4 for Agile 3+, (2) improvement in the identification of advanced fibrosis (Agile 3+) with higher sensitivity compared with FIB-4 and higher positive likelihood ratio compared with LSM in the rule in zone, and (3) overall better discrimination with the least number of patients in the indeterminate zone compared with both FIB-4 and LSM, except for Agile 3+ (14%) compared with LSM (13%).
The improvements associated with Agile 3+ and Agile 4 are particularly important, as patients with ≥ F3 and F4 are at the highest risk of developing clinical liver events and outcomes; these 2 groups were previously not sufficiently well ruled in using LSM by VCTE of FIB-4. [16] Adding these scores will increase the confidence of users that their patients have either ≥ F3 or F4 and eventually identify patients candidates for therapies more accurately in clinical practice and trigger surveillance for the occurrence of complications such as esophageal varices and HCC. Major strengths of Agile 3+ and 4 include their noninvasive combination of serum biomarkers and LSM by VCTE and performance accuracy. Through the noninvasive identification of, respectively, advanced fibrosis and cirrhosis, the Agile 3+ and 4 scores decrease the need for invasive liver biopsies. As FIB-4 and LSM by VCTE are insufficient for ruling-in advanced stages of fibrosis, the Agile 3+ and 4 scores also reduce additional healthcare testing that might have been warranted in the case of a positive FIB-4 or LSM. [7] Furthermore, the Agile 3+ and 4 scores' better discrimination with reduced number of patients in the indeterminate zone will inform next steps and aid clinical decision-making. Finally, the Agile 3+ and 4 scores use dual score cutoffs that may be applied to identify advanced fibrosis and cirrhosis, respectively, in the clinical trials and clinical setting with high utility and ease.
This study has several limitations. This study is a retrospective analysis. However, the overall study cohort consisted of 4 well-characterized study populations that not only originate from multiple tertiary care centers that follow standardized criteria for performing noninvasive testing and liver biopsy but also included the entire spectrum of NAFLD. Furthermore, the study cohort was large despite its adherence to stringent study inclusion requirements including laboratory tests liver biopsy, and LSM by VCTE within 6 months. Second, the prevalence rates for advanced fibrosis (46%) and cirrhosis (26%) are higher than those in the general community, but these high prevalence rates empower robust analysis of the performances of the Agile 3+ and 4 scores in comparison to FIB-4 and LSM. Third, although histological readings were not completed centrally, readings were performed by experienced hepatopathologists at each center. Fourth, clinical uptake of the Agile 3+ and 4 scores may be limited by availability of VCTE that requires investing in training personnel and device supply. Still, VCTE remains more cost-effective and risk free than similar imaging modalities such as CT or MRI. [17][18][19] Indeed, VCTE has already been implemented by many experts and society guidelines as an important step for assessing disease severity in NAFLD patients. [20,21] In addition, the constitutive demographic data (age, sex, and presence of type 2 diabetes) and serum biomarkers (aspartate aminotransferase, alanine transaminase, and platelets) used in Agile 3+ and 4 are incorporated in the standard examination of any liver disease. The Agile 3+ and 4 scores have been included into online calculators with high clinical utility.
In summary, introducing the noninvasive Agile 3+ and 4 scores is important given its improved discrimination with decreased indeterminate rate and increased accuracy in properly ruling in and ruling out patients with late stages of fibrosis. By successfully identifying advanced fibrosis and cirrhosis, respectively, the Agile 3+ and 4 scores will aid clinicians in targeting those who are at higher risk for liver-related mortality and may benefit from therapeutic intervention and surveillance for the development of end-stage liver disease complications.

AUTHOR CONTRIBUTIONS
Mazen Noureddin: participated in the design of the study, interpreted the data, and critically revised the manuscript for important intellectual content. Edward Mena, Raj Vuppalanchi, Niharika Samala, Micaela Wong, Fabiana Pacheco, Prido Polanco, Celine Sakkal, Ani Antaramian, Devon Chang, Nabil Noureddin, Anita Kohli, Stephen A. Harrison, Samer Gwarieh3, and Naim Alkhouri: interpreted the data and critically revised the manuscript for important intellectual content. Emily Truong: participated in the design of the study, interpreted the data, and drafted the manuscript.

FUNDING INFORMATION
EchoSens financially supported this project in the later stages.